NHE3 mRNA Expression Research Articles

Altered colonocyte differentiation may lead to decreased electrolyte and fluid absorption in the ileocolon of IBD patients by affecting the expression pattern of the responsible ion transporters

Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases (IBD) characterized by disturbed homeostasis of the immune system, the intestinal mucosa and the microbiome, causing ‘leaky’ intestinal epithelial barrier with altered fluid and electrolyte absorption leading to diarrhea. Fluid absorption in the ileocolonic region is predominantly mediated by the Cl-/HCO3 - exchanger SLC26A3 (DRA) and the Na+/H+ exchanger SLC9A3 (NHE3), while in the distal colon, the epithelial Na+ channel (ENaC) also plays a role. The objective of the current study is to evaluate if the downregulation of specific absorptive ion transporters or general defect of the enterocyte differentiation correlate with the degree of diarrhea and inflammation. For this reason, we have analyzed the mRNA expression and/or protein abundance of DRA, NHE3 and ENaC, as well as a panel of genes that correlate with the differentiation state of the colonic epithelium, in different segments of the ileocolon of healthy individuals and IBD patients with an acute flare or in remission. Endoscopy and histopathologic scoring and TNFα mRNA levels were used to grade inflammation, clinical scores for disease activity and diarrhea. mRNA expression and immunohistochemical staining of DRA and NHE3 in healthy colonic tissue revealed differences in the expression pattern of DRA and NHE3 along the human colon, with DRA being most abundant in the transverse and descending colon and NHE3 in the ascending colon (n=10). The reduction of DRA and NHE3 mRNA expression/abundance in the membrane correlated with the degree of inflammation as well as the diarrhea score (n=42), while in inactive disease the expression and abundance of DRA and NHE3 did not differ from healthy controls (n=13). Furthermore, significant decrease of the ENaC subunit ENaCγ expression was detected in inflamed colon sigmoideum (n=30). Since the addressed ion transporters are expressed on differentiated intestinal epithelial cells, we further investigated if their altered expression is linked to a possible defect of the epithelial differentiation program. It was found that the mRNA expression of the absorptive enterocyte differentiation markers intestinal alkaline phosphatase (iALP), villin and HES1 was significantly decreased in inflamed colon, while the expression of the proliferative marker Ki67, HOPX, BM1 and Claudin2 was significantly increased. However, the expression of the stem cell marker LGR5 was not altered (n=30). The results showed a disrupted transcriptional regulation of the epithelial ion transporters DRA, NHE3 and ENaCγ in the inflamed epithelium of IBD patients, which will lead to their reduced function and cause electrolyte/water accumulation leading to diarrhea. The decreased expression of the epithelial ion transporters could be a consequence of the altered enterocyte differentiation of the inflamed epithelium, most likely due to signaling pathways triggered by the proinflamatory cytokines. Grant Support: DFG SE 460/21-1 and 22-1, and VW Z1953. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vitamin D regulates transepithelial acid secretion in zebrafish (Danio rerio) larvae

Maintenance of an acid-base balance is essential for normal physiological processes in vertebrates. Freshwater fishes live in an aquatic environment with variable pH, and their buffering capacity for acid-base balance in body fluids is weak. Thus, after acid exposure, fishes secrete excess acid to prevent internal acidosis. Acid-secreting ionocytes present in the adult gills and embryonic skin are primarily responsible for acid secretion, and H+-ATPase and Na+/H+ exchanger 3 (NHE3) are the two main transporters responsible for apical acid secretion. Vitamin D is a well-known hormone involved in the maintenance of Ca2+ homeostasis and is suggested to be involved in acid-base regulation by modulating the activity and/or mRNA expression of NHE3 in mammalian models. It remains unclear whether vitamin D is involved in acid secretion in fishes. The aim of the present study was to use zebrafish as a model to determine whether vitamin D and its receptors influence acid secretion. Our results indicated that the levels of 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3), the bioactive vitamin D, were significantly increased in 3 days post-fertilization zebrafish larvae after exposure to acidic freshwater (AFW, pH 4.0). Exogenous 1α,25(OH)2D3 (20 μg/L) incubation substantially enhanced the mRNA expression of acid-secreting transporters and acid secretion at the skin of the entire body and each H+-ATPase-rich cell (HRC), a type of acid-secreting ionocyte. Furthermore, the expression of vitamin D receptors (VDRs) was identified in HRCs of zebrafish. When both VDRa and VDRb were knocked down, acid secretion and the mRNA expression of acid-secreting transporters were significantly decreased. Moreover, double knockdown of VDRa/b prevented the increase in acid secretion induced by AFW and 1α,25(OH)2D3 treatment. This study is the first to indicate that vitamin D is involved in acid secretion in fish.

Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth

Dysregulation of intra- and extracellular pH in cancer contributes to extracellular matrix remodeling, favors cell migration, proliferation, and metastasis. Although the primary attention has been focused on the role of the ubiquitous Na+/H+ exchanger isoform NHE1, the role of NHE3, the predominant apical isoform in colonic surface epithelium in the pathogenesis of colon cancer has not been investigated. Here, we show that NHE3 mRNA expression is significantly reduced in colorectal cancer patients and that low NHE3 expression is associated with poorer survival. Deletion of NHE3 in ApcMin mice evaluated at 15 weeks of age (significant mortality was observed beyond this time) led to lower body weights, increased mucosal inflammation, increased colonic tumor numbers, evidence of enhanced DNA damage in tumor surface epithelium, and to significant alteration in the gut microbiota. In the absence of the inflammatory and microbial pressors, ca. 70% knockdown of NHE3 expression in SK-CO15 cells led to reduced intracellular pH, elevated apical pH, dramatic differences in their transcriptomic profile, increased susceptibility to DNA damage, increased proliferation, decreased apoptosis and reduced adhesion to extracellular matrix proteins. Our findings suggest that loss of NHE3 in the surface epithelium of colonic tumors has profound consequences for cancer progression and behavior.

Rosmarinic Acid, as an NHE1 Activator, Decreases Skin Surface pH and Improves the Skin Barrier Function.

Stratum corneum (SC) pH regulates skin barrier functions and elevated SC pH is an important factor in various inflammatory skin diseases. Acidic topical formulas have emerged as treatments for impaired skin barriers. Sodium proton exchanger 1 (NHE1) is an important factor in SC acidification. We investigated whether topical applications containing an NHE1 activator could improve skin barrier functions. We screened plant extracts to identify NHE1 activators in vitro and found Melissa officinalis leaf extract. Rosmarinic acid, a component of Melissa officinalis leaf extract, significantly increased NHE1 mRNA expression levels and NHE1 production. Immunofluorescence staining of NHE1 in 3D-cultured skin revealed greater upregulation of NHE1 expression by NHE1 activator cream, compared to vehicle cream. Epidermal lipid analysis revealed that the ceramide level was significantly higher upon application of the NHE1 activator cream on 3D-cultured skin, compared to application of a vehicle cream. In a clinical study of 50–60-year-old adult females (n = 21), application of the NHE1 activator-containing cream significantly improved skin barrier functions by reducing skin surface pH and transepidermal water loss and increasing skin hydration, compared to patients who applied vehicle cream and those receiving no treatment. Thus, creams containing NHE1 activators, such as rosmarinic acid, could help maintain or recover skin barrier functions.

Protective Effect of Coriander (Coriandrum sativum L.) on High-Fructose and High-Salt Diet-Induced Hypertension: Relevant to Improvement of Renal and Intestinal Function.

Hypertension has become a leading cardiovascular risk factor worldwide. In this study, we explored the salutary effects and relevant mechanisms of coriander (Coriandrum sativum L.), an herbal plant with culinary and medicinal values, on high-fructose and high-salt diet (HFSD)-induced hypertension in SD rats. Our results showed that oral administration of coriander (1.0 or 2.0 g/kg·bw) effectively attenuated HFSD-induced elevation of systolic blood pressure, diastolic blood pressure, and mean arterial pressure. Coriander also increased the serum levels of vasodilator factors (PGI2, NO, and eNOS), decreased Na+ retention and serum uric acid (UA) level, and ameliorated glucolipid profiles. qPCR results revealed that coriander downregulated the mRNA expression of NHE3, a Na+/H+ exchanger responsible for Na+ absorption, in kidney and small intestine. 16S rDNA sequencing showed that coriander altered the gut microbiota composition with the beneficial bacteria Bifidobacterium and Oscillibacter significantly enriched. Correlation analysis indicated that the abundance of Bifidobacterium was evidently correlated with levels of NHE3, NO, eNOS, and UA. LC-MS/MS analysis revealed that coriander contained a variety of flavonoids including rutin and quercetin. Conclusively, long-term consumption of coriander may ameliorate HFSD-induced hypertension by mitigating HFSD-caused abnormal changes in vascular endothelial function, renal and intestinal sodium absorption, glucolipid homeostasis, and gut microbiota in rats.

Estrogen-related receptor γ2 controls NaCl uptake to maintain ionic homeostasis.

Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that Errγ2 may play a crucial role in ion uptake. In situ hybridization showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na+/Cl- transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na+/Cl- cotransporter (Ncc), the number of Ncc ionocytes, Na+/Cl- influxes of ionocytes, and whole-body Na+/Cl- contents. In FW with low Na+ and low Cl-, the expression levels of mRNA for Na+/H+ exchanger 3 (Nhe3) and Ncc were both decreased in Errγ2 morphants. Treating embryos with DY131, an agonist of Errγ, increased the whole-body Na+/Cl- contents and ncc mRNA expression in Errγ2 morphants. As such, medaka Errγ2 may control Na+/Cl- uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.

Chemical composition and anti-inflammatory activity of n-butanol extract of Piper sarmentosum Roxb. In the intestinal porcine epithelial cells (IPEC-J2)

Ethnopharmacological relevancePiper sarmentosum Roxb. (PS) is a terrestrial herb primarily distributed in tropical and subtropical regions of Asia. It is widely used in folk medicine in certain countries of Southeast Asia for the treatment of fever, toothache, coughing and pleurisy, which showed the anti-inflammatory activity of PS. Aim of the studyThis study aimed to investigate the chemical constituents and the molecular mechanism and related metabolic pathway by which n-butanol extract of PS (PSE-NB) exerts its anti-inflammatory effects. Materials and methodsChemical constituents of PSE-NB was analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique. Anti-inflammatory effects of PSE-NB were investigated in lipopolysaccharide (LPS)-induced IPEC-J2 cells. ResultsIn total, 218 compounds, including 94 alkaloids and 26 phenolics were tentatively identified, which indicating alkaloids and phenolics were the main constituents of PSE-NB. In addition, the current cell experiment in vitro showed that PSE-NB (10–500 μg/mL) pre-treatment before LPS stimulation significantly decreased mRNA expression of IL-1β, IL-6 and TNF-α in IPEC-J2 cells compared with LPS treatment (p < 0.05). PSE-NB improved mRNA expression of tight junction proteins (ZO-1 and Occludin) and NHE3, which were reduced by LPS stimulation (p < 0.05). Moreover, PSE-NB (10 μg/mL) alleviated LPS-induced protein expression of p65 and p-p65 (p < 0.05), and reduced p65 translocation into the nucleus induced by LPS. At the same time, metabolic pathway analysis indicated that PSE-NB exerts anti-inflammatory effects mainly via augmentation of methionine metabolism in IPEC-J2 cells. ConclusionsTaken together, the results suggested that alkaloids and phenolics were the main constituents in PSE-NB. PSE-NB might attenuate LPS-induced inflammatory responses in IPEC-J2 cells by regulating NF-κB signaling pathway and intracellular metabolic pattern.

Proximal tubule albumin uptake – potential role for endothelin system in sickle cell disease mice

Elevated plasma endothelin‐1 (ET‐1) reported in sickle cell disease (SCD) patients correlate with microalbuminuria, a major mortality risk factor in SCD. ET‐1 contributes to glomerular and tubular injury in SCD, as evidenced by increased glomerular permeability to albumin and urinary excretion of tubular injury markers. Although selective ETA receptor antagonism reduces albuminuria in humanized sickle cell (HbSS) mice, the mechanism of this action remains unclear. The aim of the study was to determine whether selective ETA receptor antagonism prevents albuminuria by preserving the expression of tubular albumin‐associated transporters in proximal tubule (PT) cells. Male C57BL/6 or HbSS and genetic control (HbAA) mice were used to determine the effect of ET‐1 on the expression of proximal tubule albumin trafficking mediators, such as megalin and NHE‐3. Exposure of primary mouse PT cells to ET‐1 (50nM) for 8h decreased megalin (53% reduction) and doubled NHE‐3 expression. Pre‐treatment with the ETA antagonist, BQ123 (1 μM), preserved megalin expression and had no effect on NHE‐3. Moreover, selective ETB receptor blockade (BQ788, 1 μM) prevented ET‐1‐mediated increase in NHE‐3 expression. Primary PT cells isolated from HbSS mice showed decreased megalin mRNA expression as well as protein abundance relative to HbAA PT controls. Ten‐week treatment with selective ETA receptor antagonist (10mg/kg/day) preserved expression of megalin at control levels. There were no differences in NHE‐3 mRNA expression in HbSS PT cells regardless the treatment. Interestingly, PT cells from HbSS cultured with HbSS plasma and ET‐1 (10nM) had decreased NHE‐3 protein abundance compared to non‐treated cells. These results potentially uncover a novel role for ET‐1 in PT albumin handling and suggest that PT ET‐1 receptor signaling contributes to albuminuria in SCD.Support or Funding InformationSupport provided by an NIH U01 HL117684 to D.M.P, AHA 19CDA34660073 and NIH‐NHLBI K99HL144817 to M.K.

Impairment of electroneutral Na+transport and associated downregulation of NHE3 contributes to the development of diarrhea following in vivo challenge withBrachyspiraspp.

The effect of Brachyspira hyodysenteriae and Brachyspira hampsonii spirochetosis on Na+ transport was assessed in the colon to determine its contribution to diarrheal disease in pigs following experimental infection. Electrogenic and electroneutral Na+ absorption was assessed in Ussing chambers by radiolabeled 22Na flux and pharmacological inhibitory studies. Basal radiolabeled 22Na flux experiments revealed that mucosal-to-serosal flux (Jms) was significantly impaired in B. hyodysenteriae and B. hampsonii-diseased pigs. Inhibition of epithelial sodium channel via amiloride did not significantly reduce electrogenic short-circuit current (Isc) in the proximal, apex, and distal colonic segments of diseased pigs over control pigs, suggesting that a loss of electroneutral Na+ absorption is responsible for diarrheal development. These findings were further supported by significant downregulation of Na+/H+ exchanger (NHE1, NHE2, and NHE3) mRNA expression in the proximal, apex, and distal colonic segments paired with decreased protein expression of the critical NHE3 isoform. The decrease in NHE3 mRNA expression appears not to be attributed to the host's cytokine response as human IL-1α did not modify NHE3 mRNA expression in Caco-2 cells. However, a whole cell B. hampsonii lysate significantly downregulated NHE3 mRNA expression and significantly increased p38 phosphorylation in Caco-2 cells. Together these findings provide a likely mechanism for the spirochete-induced malabsorptive diarrhea, indicated by a decrease in electroneutral Na+ absorption in the porcine colon due to Brachyspira's ability to inhibit NHE3 transcription, resulting in diarrheal disease.NEW & NOTEWORTHY This research demonstrates that diarrheal disease caused by two infectious spirochete spp. is a result of impaired electroneutral Na+ absorption via Na+/H+ exchanger 3 (NHE3) in the porcine colon. Our findings suggest that the decrease in NHE3 mRNA and protein is not likely a result of the host's cytokine response. Rather, it appears that these two Brachyspira spp. directly inhibit the transcription and translation of NHE3, resulting in the development of diarrhea.

Effect of salinity and temperature on the expression of genes involved in branchial ion transport processes in European sea bass.

The responses of European sea bass to temperature increase and salinity decrease were investigated measuring mRNA expression levels of main genes involved in ion transport. Juvenile fish were pre-acclimated to seawater (SW) at 18 °C (temperate) or 24 °C (warm) for two weeks and then transferred for two weeks to either fresh water (FW) or SW at the respective temperature. Unlike temperate conditions, there is no change in Na+/K+-ATPase α1a (nka α1a) and Na+/H+ exchanger 3 (nhe3) mRNA expression following FW transfer in warm conditions. This is linked to the high expression of these genes in warm SW compared to temperate SW. Na+/Cl--cotransporter (ncc2a) expression however is increased following FW transfer in temperate and warm conditions. Main transporters involved in ion excretion (Na+/K+/2Cl--1 cotransporter, nkcc1 and cystic fibrosis transmembrane conductance regulator, cftr) as well as nitrogen excretion (Rh-glycoproteins, rhcg1 and rhbg) and acid-base regulation (V-H+-ATPase, vha-a and b) are highly expressed in SW warm conditions vs FW warm. Overall, our results suggest a higher activation of ion transport processes in warm conditions and more strikingly in SW. This is linked to a strong interplay between diverse ion transporters in order to coordinate physiological responses at the gill level.

Testosterone Reduces mRNA Expression of Renal Sodium Transporters but does not Affect Blood Pressure During a Ten‐Day Angiotensin II Infusion Period in Castrated Mice

Sex differences in blood pressure regulation are well known and it is widely accepted that premenopausal females are protected from hypertension and cardiovascular disease compared to age‐matched males. Despite extensive investigation, the effects of sex steroids on blood pressure and renal sodium handling remain debated. The purpose of this study was to determine the effects of testosterone administration on angiotensin II (Ang II) – induced increase in blood pressure and other physiological effects in castrated mice. Four‐week‐old CD‐1 castrated mice were obtained from Envigo Inc. (Indianapolis, IN) and placed in metabolic cages for a five‐day baseline period followed by implantation of an Alzet osmotic pump containing either vehicle or Ang II (1μg/kg/min) and either a placebo or 2.5 mg testosterone‐acetate pellet (Innovative Research of America, Sarasota, FL). Measurements were recorded daily in the five‐day baseline and ten‐day post‐implantation periods in three groups of mice: vehicle‐placebo (V‐P), Ang II‐placebo (Ang II‐P), and Ang II‐testosterone (Ang II‐T) (n=4/group). Mice consumed standard chow and water ad libitum throughout the study. Systolic blood pressure (SBP, mmHg) was determined daily with the tail‐cuff technique (Kent Scientific, Torrington, CT). At the end of the study, mice were sacrificed, kidneys were removed, and cortical tissue samples were processed for real‐time qtPCR to compare mRNA expression in fold difference (FD) of key sodium transporters. Ang II significantly increased blood pressure. The delta SBP (baseline vs Ang II periods) was not different between the Ang II‐P and Ang II‐T groups: (27.0 ± 4.5 vs 31.1 ± 5.9, respectively), and these values were not different from the delta SBP (27.7 ± 2.7) in intact male (IM) mice under the same Ang II conditions determined in a previous study (Rouch, A. el al. FASEB J, 2018, A 904.6). Ang II administration significantly increased water intake and urine flow rate compared to baseline periods in Ang II‐P &amp; Ang II‐T mice with no differences between those two groups, and there was no difference among groups with respect to sodium excretion or sodium balance. Interestingly, renal cortical tissue samples from Ang II‐P mice had higher mRNA expression of ENaC (a,b, &amp; g), NKCC, NCC, and NHE3 compared to those from Ang II‐T mice (FD: 0.7–2.5, p &lt; 0.05). Tissue samples from V‐P mice expressed higher mRNA levels of ENaC (b&amp; g), Na‐K ATPase‐a1, NCC, and NHE2 compared to samples from IM mice (FD: 1.7–2.5, p &lt; 0.05). We conclude that testosterone does not affect the ten‐day Ang II‐induced increase in blood pressure in the CD‐1 mouse. Testosterone reduces mRNA expression of key renal sodium transporters. Physiological consequences of T‐induced changes in mRNA expression remain to be determined.Support or Funding InformationThis project was supported by the National Institute of General Medical Sciences of National Institutes of Health through Grant Number 8P20GM103447.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decreased expression of Na+-H+ exchanger isoforms 1 and 3 in denervated spontaneously hypertensive rat kidney

ABSTRACTTo determine whether the sympathetic nerve plays a role in the regulation of Na+-H+ exchange (NHE) in the kidney of spontaneously hypertensive rats (SHR), we investigated the expression of NHE and NHE regulatory protein family (NHERF) in the denervated kidneys compared with intact kidneys. Twelve-week-old male SHR and age-matched Wistar Kyoto (WKY) rats were used. SHR were randomly assigned to the renal denervated (RDNX, n = 8) or Sham (n = 8) groups. The protein and mRNA expression of NHE1, NHE3, NHERF1 and NHERF2 were assessed in the kidney of the groups. Following the renal denervation, immunohistochemistry and western blot analysis showed that NHE1 and NHE3 protein were significantly decreased in the kidney compared with Sham group. NHERF1 protein expression was significantly increased in RDNX compared with Sham group, whereas NHERF2 protein expression was significantly decreased after renal denervation. Similar results were observed at the mRNA level of NHE1, NHE3, NHERF1 and NHERF2 expression. The present findings suggest that the renal sympathetic nervous system plays a role in the regulation of NHE1 and NHE3 in the kidney of SHR, and NHERF1 may be involved in the expression of NHE3 in the kidney of SHR.

Rho Kinase and Na+/H+ Exchanger Mediate Endothelin‐1 Induced Pulmonary Arterial Smooth Muscle Cell Proliferation and Migration

ObjectiveRemodeling of the pulmonary vasculature, involving increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a key component of the pathology underlying pulmonary hypertension (PH). Endothelin‐1 (ET‐1) is known to be an important contributor to both pulmonary vasoconstriction and remodeling in PH. We aimed to determine whether ET‐1 modulates PASMC function via a pathway involving Rho kinase (ROCK) and the Na+/H+ exchanger (NHE).MethodsPASMCs were isolated from distal pulmonary arteries of male C57/B6 mice as well as male Wistar rats and expanded in culture. To measure NHE activity, cells were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH (pHi) following acidification by ammonium pulse challenge was measured via fluorescence microscopy. NHE1 mRNA expression was measured via qRT‐PCR and protein expression was measured via immunoblot. Proliferation was assessed via ELISA for BrdU incorporation over 24 hr. Migration was measured over 24 hr using Transwell filters with 8 μm pores. Cells were treated with 10−8 M ET‐1 versus vehicle for 24–48 hr. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride. ROCK was inhibited with 10 μM Y‐27632.ResultsET‐1 treatment, compared to vehicle, resulted in increased resting pHi and NHE activity in PASMCs. ET‐1 did not result in increased NHE1 mRNA or protein expression. ROCK inhibition prevented ET‐1‐induced NHE activation. PASMC proliferation and migration were both increased by ET‐1 treatment, compared to vehicle. ROCK inhibition attenuated PASMC proliferation and migration in the presence of ET‐1. Similarly, NHE inhibition also attenuated ET‐1‐induced PASMC proliferation and migration.ConclusionsET‐1 results in increased PASMC proliferation and migration via a pathway involving ROCK and NHE activation. This suggests a novel pathway which may contribute to vascular remodeling in PH.Support or Funding InformationNIH K08HL133475 &amp; R01HL073859This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression.

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 μM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

Effects of Na+ -H+ exchanger 1 knockdown on protein expression levels of ATP binding cassette transporter A1 and cholesterol efflux in hypoxic RAW264.7 cells

Objective To explore the effects of Na+ -H+ exchanger 1(NHE1) knockdown on ATP binding cassette transporter A1 (ABCA1) protein expression levels and cholesterol efflux in the hypoxic RAW264.7 cells. Methods The RAW264.7 cells were infected with lentiviral vectors expressing shRNA specific for NHE1(siNHE1) or scramble RNA (siNC). The expression of NHE1 at mRNA or protein level was detected by qRT-PCR and Western blotting respectively in the infected cells after 24 h in a hypoxia condition.In the meantime, the methods of SNARF-1, Fluo-4 NW andSuc-LLVY-aminoluciferin were employed to determine NHE1 activity, intracellular Ca2+ ([Ca2+ ]i) and calpain activity, respectively.Furthermore, ABCA1 protein levels were detected by Western blotting in the 24 h hypoxic cells.In parallel, the intracellular cholesterol content and cholesterol efflux were analyzed by the methods of combined enzymatic HLPC and 3H-cholesterol. Results The hypoxia condition versus the normoxia condition up-regulated NHE1 mRNA and protein expression level and activity by 2.48 folds, 1.28 folds and 61.96% (all P<0.05), and increased[Ca2+ ]i and calpain activity by 4.51 folds and 2.41 folds(all P<0.05). Whereas the NHE1 mRNA and protein expression and activity at the presence of hypoxia were inhibited by siNHE1 with the inhibition ratio of 84.95%, 60.75% and 66.44%, respectively (all P<0.05) and[Ca2+ ]i and calpain activity were reduced by 59.23% and 54.66% (P<0.05). Furthermore, the ABCA1 protein level was 61.67% lower in the hypoxic cells than in the normoxic cells (P<0.05), and siNHE1 was increased by 56.52%after treatment of Hypoxia.Hypoxia elevated intracellular total cholesterol and cholesterol ester by 74.57% and 101.81% (all P<0.05). Treatment with siNHE1 in the hypoxia condition can reduce total cholesterol and cholesterol ester by 34.24%及49.66%(all P<0.05). Hypoxia reduced the cholesterol efflux by 34.79%(P<0.05), which were partially reversed by siNHE1. Conclusions NHE1 might play an important role in hypoxia-induced ABCA1 protein attenuation and reverse cholesterol transport dysfunction through[Ca2+ ]i/calpain pathway. Key words: ATP binding cassette transporter A1; Na+ -H+ exchanger 1; Anoxia; Cholesterol

Modulation of oxidative and glycolytic skeletal muscle fibers Na+/H+ exchanger1 (NHE1) and Na+/HCO3- co-transporter1 (NBC1) genes and proteins expression in type 2 diabetic rat (Streptozotocin + high fat diet) following long term endurance training.

Diabetes is known to alter both oxidative and glycolytic pathways in a fiber type-dependent manner. The aim of present study was to investigate the effects of endurance training on muscle NHE1 and NBC1 genes and proteins expression in type 2 diabetic rats. Male wistar rats (n=30), 4 weeks old and 95.7±10.8g, were randomly selected and divided into control, diabetic without training and diabetic with training groups. Diabetes was induced by injection of low dose of streptotozin and feeding with high-fat diet. The Endurance training was performed for 7 weeks that started with relatively low speed and duration of 20 m min-1 for 20 min in the first week and gradually reached to 30 m min-1 for 35min in the last week. NHE1 and NBC1 genes and proteins expression were determined by Real time-PCR and western blotting techniques, respectively, in Soleus as an oxidative and EDL (Extensor digitorum longus) as a glycolytic muscle preparation. NHE1 mRNA and protein expression reduced significantly in EDL and Soleus in the diabetic without training group compared with the control group. However, reduction in the expression of NBC1 gene and protein in the diabetic without training group compared to controls did not significant. Endurance training increased NHE1 and NBC1 genes and proteins expression in both EDL and Soleus in the diabetic training group compared to control groups. In conclusion, endurance training may improve the capacity of pHi regulation in muscles by lactate-independent pathway.

Loss of Myosin Vb in Intestinal Enterocytes Results in Aberrant Expression of Key Apical Transporters in vitro

BackgroundInactivating mutations in myosin Vb (MYO5B) result in Microvillus Inclusion Disease (MVID). MVID is a rare form of congenital diarrhea characterized by morphological intestinal enterocyte abnormalities and aberrations in the localization of brush border proteins and enzymes. Despite recent attention on MVID, the origin of the diarrheal disorder remains unclear. Our lab has generated a mouse model of MVID with a germline deletion of MYO5B (MYO5B KO). These mice rapidly develop diarrhea, have failure to thrive and demonstrate numerous microvillus inclusions in the intestinal enterocytes.HypothesisWe hypothesize that loss of MYO5B results in the improper localization of key apical membrane components away from the brush border resulting in inadequate absorption of nutrients and water.MethodsThe 3D culture of intestinal epithelial organoids, known as enteroids, allows for the in vitro propagation of all of the intestinal cell types. Enteroids were used as a simplified model to study epithelial alterations that result from loss of MYO5B. Enteroids were generated from the duodenum of neonatal mice with a germline deletion of MYO5B (MYO5B KO) and heterozygous or wildtype littermates (control). Enteroids were differentiated for 3–5 days by withdrawal of Wnt3A and analyzed for the localization of apical transporters by immunostaining. Quantitative real time PCR was used to determine the relative gene expression of transporters in MYO5B KO and control derived enteroids.ResultsF‐actin staining using phalloidin conjugates showed a mature brush border in enteroids derived from neonatal control mice. MYO5B KO derived enteroids also showed apical F‐actin staining, but F‐actin and ezrin positive inclusions were observed within enterocytes. These inclusions closely resembled inclusions seen in the intestinal tissue of MYO5B KO mice. SGLT1 and DPPIV were expressed on the apical membrane of control enteroids. In contrast, MYO5B KO derived enteroids had diffuse, subapical SGLT1 and DPPIV expression. NHE3 immunofluorescence showed apical expression of NHE3 in control enteroids and decreased expression in MYO5B KO enteroids. qRT‐PCR data showed a significant increase in SGLT1 and NHE3 mRNA expression in MYO5B KO enteroids compared to control enteroids. This could indicate that SGLT1 and NHE3 mRNA may be up‐regulated in an attempt to compensate for degradation or improper localization. Enteroids generated from MYO5B KO mice also showed a significant increase in proliferation compared to control derived enteroids, consistent with an increase in proliferation observed in the intestinal crypts of MYO5B KO mice.ConclusionsCollectively these data suggest that enteroids from MYO5B KO mice maintain the aberrant enterocyte characteristics seen in vivo and thus will be a good model for elucidating the mechanism by which loss of MYO5B results in untreatable diarrhea. The enteroid model of loss of MYO5B demonstrates intestinal epithelial changes that suggest that the improper localization of apical transporters is responsible for the diarrheal pathology seen in patients with MVID.Support or Funding InformationNIDDK Grant RO1 DK048370 and T32 Training Grant 2T32CA106183‐11.

Salt supplementation ameliorates developmental kidney defects in COX-2-/- mice.

Deficiency of cyclooxygenase-2 (COX-2) activity in the early postnatal period causes impairment of kidney development leading to kidney insufficiency. We hypothesize that impaired NaCl reabsorption during the first days of life is a substantial cause for nephrogenic defects observed in COX-2-/- mice and that salt supplementation corrects these defects. Daily injections of NaCl (0.8 mg·g-1·day-1) for the first 10 days after birth ameliorated impaired kidney development in COX-2-/- pups resulting in an increase in glomerular size and fewer immature superficial glomeruli. However, impaired renal subcortical growth was not corrected. Increasing renal tubular flow by volume load or injections of KCl did not relieve the renal histomorphological damage. Administration of torsemide and spironolactone also affected nephrogenesis resulting in diminished glomeruli and cortical thinning. Treatment of COX-2-/- pups with NaCl/DOCA caused a stronger mitigation of glomerular size and induced a slight but significant growth of cortical tissue mass. After birth, renal mRNA expression of NHE3, NKCC2, ROMK, NCCT, ENaC, and Na+/K+-ATPase increased relative to postnatal day 2 in wild-type mice. However, in COX-2-/- mice, a significantly lower expression was observed for NCCT, whereas NaCl/DOCA treatment significantly increased NHE3 and ROMK expression. Long-term effects of postnatal NaCl/DOCA injections indicate improved kidney function with normalization of pathologically enhanced creatinine and urea plasma levels; also, albumin excretion was observed. In summary, we present evidence that salt supplementation during the COX-2-dependent time frame of nephrogenesis partly reverses renal morphological defects in COX-2-/- mice and improves kidney function.

Distinct protein and mRNA kinetics of skeletal muscle proton transporters following exercise can influence interpretation of adaptations to training.

What is the central question of this study? Following a training intervention, how is the interpretation of adaptations in skeletal muscle H+ transporters influenced by biopsy timing in the context of individual protein and mRNA kinetics after the final exercise bout? What is the main finding and its importance? We show that distinct postexercise protein and mRNA kinetics for monocarboxylate transporter1/4 and sodium-hydrogen exchanger1 indicate that timing of a single end-point biopsy after a training intervention can influence the inferences made. Furthermore, we found the intrasubject, intersample variability of the muscle buffer capacity titration assay to be greater than the typical training effect. In order to gain a better understanding of training-induced adaptations in skeletal muscle pH regulation, in this study we measured protein and mRNA kinetics of proton (H+ ) transporters for 72h following a bout of high-intensity interval exercise (HIIE), conducted after 4 weeks of similar training. We also assayed muscle buffer capacity (βm) by a titration technique (βmin vitro ) over the same period. Sixteen active men cycled for seven bouts of 2min at ∼80% of peak aerobic power, interspersed with 1min rest. Compared with the first 9h postexercise, monocarboxylate transporter (MCT)1 protein content was ∼1.3-fold greater 24-72h post-HIIE, whereas there was no such change in MCT4 protein content. Conversely, MCT1 and MCT4 mRNA expression progressively decreased 9-72h post-HIIE. Sodium-hydrogen exchanger1 (NHE1) protein content was lower 9h post-HIIE (∼0.8-fold) compared with every other postexercise time point, but NHE1 mRNA expression was 2.2 to 2.9-fold greater 24-72h post-HIIE, compared with the first 24h post-HIIE. Furthermore, we determined the intrasubject, intersample variability (11.5%) of βmin vitro for resting samples taken on consecutive days to be greater than the typical training effect (mean 6%; 95% confidence limits ±4%). In conclusion, the delay in steady-state protein turnover should inform biopsy timing in studies investigating the response to training of the H+ transport proteins, whereas the temporal resolution provided by single time points has been shown to be of limited epistemological value for their corresponding mRNA expression. Finally, our data cast doubt on the ecological validity of the βmin vitro assay for measuring true changes in βm.

Phosphorylation and subcellular localization of Na+/H+ exchanger isoform 3 (NHE3) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones.

Na+/H+ exchanger isoform 3 (NHE3) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE3 in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE3 mRNA expression. NHE3 protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE3 mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE3 protein. However, a higher level of NHE3 phosphorylated at serine-552 (P-NHE3) was seen on the lithogenic diet. In immunofluorescence studies, NHE3 protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE3 strongly corroborates an exchanger trafficking mechanism in NHE3 activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE3 following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function.